The use of spectrophotometers to measure the light absorption characteristics of sample materials is well known. Indeed, the basic principles involved are relatively simple. A beam of light, whose characteristics are known, is directed through the sample material and the light that emerges is analyzed to determine which wavelengths of the original beam were absorbed, or otherwise affected, by the sample material. Based on differences between the incident light and the transmitted light, certain characteristics of the sample material can be determined. Many variables are involved, however, that can make a spectrophotometric measurement quite complex. In sum, these complexities arise from the fact that the sensitivity and accuracy of a measurement rely on the ability of the spectrophotometer to measure the light which is absorbed by the samples.
Analytically, a spectrophotometric analysis relies on a known relationship of the variables involved. Specifically, in a standard spectrophotometric measurement, the amount of light transmitted through a test cuvette is measured and the percent of transmitted light is related to the material in the cuvette by the following relationship: EQU I.sub.t (.lambda.)=I(.lambda.) 10.sup.-OD
where I(.lambda.) and I.sub.t (.lambda.) are respectively the input and transmitted intensities, and the optical density, OD, is given by: EQU OD=a(.lambda.)L C
where a(.lambda.) is the absorptivity of the material as a function of .lambda., L is the optical path length, and C is the concentration. From the above, it will be easily appreciated that the output intensity I.sub.t (.lambda.) is directly proportional to the input intensity I(.lambda.). Therefore, it is clearly necessary to have an input intensity that is sufficient to give an output intensity which can be effectively used for analysis and measurement of the sample material. Further, the efficacy of the measurement will also be enhanced if the concentration of the sample material is increased. Thus, for spectrophotometric analysis it is desirable to have a light input of high intensity, and have a highly concentrated sample in solution. There is a problem, however, when low concentration solutions of sample material are available in only very small quantities (e.g. 0.5 to 50 micrograms/microliter).
To be effective for spectroscopic measurements, test cuvettes for holding the sample material must be completely filled. This typically requires a substantial amount of sample material. Consequently, when only a small amount of the sample material is effectively available for testing, presently available test cuvettes (e.g. 12.5 mm.times.12.5 mm cuvette) are inadequate because of their relatively large size. Merely reducing the size of the cuvette is not the answer. This is so because, with a size reduction of the cuvette there is also a reduction in the amount of sample material through which light can pass. Consequently, the intensity of the light passing through the sample material is reduced and the sensitivity and accuracy of the measurement is compromised.
The present invention recognizes that it is possible to take spectrophotometric measurements of very small quantities of a sample material, even where there is a relatively low concentration of the material in solution. The present invention recognizes that this can be done by properly focusing collimated light onto the sample material to obtain sufficiently high input light intensities for the desired measurements. Further, the present invention recognizes that this focusing can be accomplished by a device which is engageable, and operatively compatible, with presently available spectrophotometers such as a UVIKON Model 820 spectrophotometer by Kontron.
The present invention further recognizes that occasionally it is important to make spectroscopic observations of small samples at various controlled elevated temperatures. For example, for DNA material, it is known that the double strands of DNA break into two single strands (denatures) at temperatures above 70.degree. C. This denaturing of the DNA is also known to result in a significant increase in the light absorption of the sample. It is desirable to spectroscopically monitor denaturization. It is also desirable to spectroscopically monitor enzymatic and other thermally-induced reactions in small biological, as well as nonbiological, samples. For example, the progress of a process known as polymerase chain reaction (PCR), disclosed in U.S. Pat. No. 4,683,202, can be studied using spectroscopic techniques. The PCR process involves repeatedly denaturing and assembling DNA in the presence of oligonucleotide primers to amplify the DNA. In this context, assembly consists of two parts, binding of the primers to the target DNA and extension from the primer sites by the polymerization of nucleotides to form double stranded DNA. More particularly, the PCR process requires cyclically heating the DNA sample in accordance with a predetermined temperature profile schedule to raise the temperature above the denaturing temperature for a predetermined dwell time and the reducing the temperature to below the denaturing temperature for a predetermined dwell time to allow the single strands to assemble from primers and nucleotides (C,G,A,T) into double strands. Unfortunately, the precise denaturation and assembly temperatures and dwell times of the PCR technique can be difficult to optimize. Optimum temperatures and dwell times are desirable in order to achieve relatively fast and efficient DNA amplification.
Importantly, as the DNA solution undergoes the cyclic denaturation/assembly of the PCR process, the light absorption characteristics of the solution change. Consequently, by observing the light absorption characteristics of the DNA solution over time, the actual progress of the PCR process can be monitored. Stated differently, the changes in the light absorption characteristics of the DNA solution can be correlated to changes in the constituent composition of the DNA solution. Consequently, the present invention recognizes that the predetermined temperature profile schedule can be changed in response to the observed changes in the light absorption characteristics of the DNA solution, in order to optimize the PCR process.
In particular, the present invention recognizes that the absorption of light at 260 nm, as observed by a spectrophotometer, reveals the amount of target/product DNA, nucleotides and oligonucleotide primer present in the DNA solution because each constituent exhibits a different absorption strength. Since there is a difference between light absorption of single stranded DNA and double stranded DNA, i.e., single stranded DNA exhibits greater absorption, the total amount of target/product DNA can be determined from the difference between absorption at the denaturation and assembly temperatures.
Additionally, light absorption can be used to monitor the quantities of other materials such as enzymes (polymeration agents), in the DNA solution which are essential to the PCR process. For example, the enzyme thermus aquatics (TAQ) is known to absorb light at 280 nm. Therefore, the amount of intact, i.e. undamaged, TAQ present in the DNA solution is determined by monitoring light absorption at 280 nm.
The present invention further recognizes that it is possible to monitor spectrophotometric and spectrofluorometric changes at biologically significant temperatures. Study of bacteria or virus growth at human body temperatures of 37.degree. C. could also be possible. In addition, nonbiological chemical reactions at temperatures elevated above room temperature can also be studied. The present invention accomplishes this by providing an apparatus which allows heating of the very small quantities of sample material in a controlled and efficient manner.
In light of the above, it is an object of the present invention to provide a micropipette adaptor for spectrophotometers which allows for spectrophotometric measurements of very small quantities of sample material in solution. Another object of the present invention is to provide a micropipette adaptor for spectrophotometers which permits recovery of the sample material after spectrophotometric measurements have been made. Yet another object of the present invention is to provide a micropipette adaptor for spectrophotometers which allows spectroscopic measurements of samples while the sample is in the process of being transferred through a micropipette. Still another object of the present invention is to provide a micropipette adaptor for spectrophotometers which provides for a high light collection efficiency to increase the sensitivity of the measurements which are made. Another object of the present invention is to provide a micropipette adaptor for spectrophotometers which allows a micropipette or other capillary sample holder to be easily installed and removed from the adaptor. Yet another object of the present invention is to provide a micropipette adaptor for spectrophotometers which provides approximately the same intensity light path length product for small samples as is provided for larger samples. Another object of the present invention is to provide a micropipette adaptor for spectrophotometers which is relatively easy to manufacture and comparatively cost-effective to operate.
Further, an object of the present invention is to provide a micropipette adaptor in which the temperature of the sample may be controlled. Another object of the present invention is to provide such a temperature-controlled micropipette adaptor which may be used in commercially available spectrophotometers. Yet another object of the present invention is to provide a temperature-controlled micropipette adaptor capable of easily attaining higher sample temperatures, and capable of maintaining predetermined temperatures for desired lengths of time. Another object of the present invention is to provide a temperature-controlled micropipette adaptor which is relatively simple and convenient to manufacture and use.